The present invention relates to a SPARC fusion protein and a method for producing the protein. The invention also relates to a pharmaceutical composition comprising the SPARC fusion protein. More specifically, the invention relates to a pharmaceutical composition for use in inhibiting nerve cell adhesion to thereby cause nerve cell migration, or for use in promoting neurite degeneration to thereby cause neuroplasticity.
SPARC (a secreted protein acidic and rich in cysteine) was originally reported as osteonectin which is a major noncollagenous protein present in bone (Thermine, J. D. et al., Cell, 26: 99-105, 1981), and has attracted attention as a functional bone-specific protein since it exhibits strong binding to collagen and hydroxyapatite and has Ca 2+binding activity. A gene encoding SPARC has been sequenced from a 43 kDa protein which is secreted by cells when adhesive cultured cells proliferate (Sage, H. et al., J. Biol. Chem., 259, 3993-4007, 1984). Later, it was also found that SPARC is identical to BM-40 isolated from the basement membrane by another group of researchers (Mann, K. et al., FEBS Lett., 218: 162172, 1987). Since then, secretion of SPARC has been observed in various cultured cells. In particular, secretion from normal fibroblasts and vascular endothelial cells is constitutive (Lane, T. F. and Sage, E. H., FASEB J., 8: 163-173, 1994).
As to functions of SPARC, it is known that when added to cultured cell systems, SPARC inhibits cell adhesion to extracellular matrix molecules in a concentration-dependent manner, and thus SPARC is called the xe2x80x9canti-adhesive proteinxe2x80x9d together with thrombospondin and tenascin (Sage, E. H. and Bornstein, P., J. Biol. Chem., 266: 14831-14834, 1991). It has been reported that this action of SPARC is not exerted by competitive inhibition of the binding between integrins and cell adhesion ligands but exerted by acting on cytoskeleton systems such as vinculin via intracellular signal transduction to thereby degrade focal adhesion plaques (Murphy-Ullrich, J. E. et al., J. Cell. Biochem., 57: 341-350, 1995). Most remarkably, SPARC reduces the production of ECM (extracellular matrix) and enhances the production of ECM-degrading proteases (Lane, T. F. and Sage, E. H., FASEB J., 8: 163-173, 1994). At the same time, SPARC is expressed highly at sites undergoing angiogenesis and reduces the secretion of fibronectin and thrombospondin in vascular endothelial cells. Thus, SPARC remodels ECM through combination of the above-described actions. In particular, it is believed that SPARC controls cell proliferation and promotes the increase of intracellular permeability in vascular endothelial cells and is essential for the expression of cell proliferating action and angiogenesis activity.
Since SPARC has various physiological activities as described above, its utility as a therapeutic is attracting attention, and clinical application of SPARC is expected.
Several attempts have been made to produce SPARC by recombinant DNA techniques using animal cells as a host cell. However, the yields were very low, and several stages of purification were needed. Thus, such methods required great labor and cost. Although there have been some attempts using Escherichia coli as a host cell, SPARC was not eluted into soluble fractions or eluted thereinto only in an extremely small amount. Besides, SPARC accumulated in inclusion bodies (which are insoluble fractions) was an inactive-type SPARC not retaining its physiological activities. A method for obtaining an active-type SPARC by appropriately folding the Sxe2x80x94S bond in the inactive-type SPARC isolated/purified from insoluble fractions has not been established yet. Even if SPARC is expressed as a fusion protein fused to glutathione-S-transferase (GST) or the like, the protein is also accumulated in inclusion bodies. Thus, any of such methods has not achieved production of SPARC in large quantities.
The above-described functions of SPARC relating to angiogenesis have been only confirmed in vascular endothelial cells. However, no reports have been made about functions of SPARC in nerve cells.
Substances which induce long-term changes in synapse function(s), such as that brought about by the administration of psychotropics like morphine, have not yet been described. Morphine and other narcotic analgesics that have a strong affinity for the opioid xcexc receptor have qualitatively almost identical pharmacological effects including: analgesic, psychological (sense of intoxication), sedative, respiration-inhibitory, vomiting, and cardiovascular effects. Thus, they are used for post-operational pain, pain of end-stage cancer, pain of cardiac infarction, etc. as an analgesic or for treating dyspnea brought about by acute pulmonary edema or acute left ventricular failure. However, though morphine exhibits a remarkable effect on pain, its use for chronic diseases causes problems of tolerance and dependence. Thus, use of morphine is restricted to the specific diseases described above. Repeated administration of morphine easily leads to tolerance, as well as physical and mental dependence. The shorter the administration interval, the quicker tolerance and dependence are formed. For example, if morphine administration is suddenly suspended or naloxone (an antagonist) is administered after the formation of physical dependence, diversified withdrawal symptoms such as trembling, anxiety, insomnia, convulsion, sweating, rhinorrhea, lacrimation, fever, rise in blood pressure, tachycardia, mydriasis, diarrhea, bellyache, vomiting, etc. appear. Since the above-described tolerance and dependence are retained for months or years, it is believed that changes are occurring in the function of synapses for a long period of time.
It is an object of the invention to generate and use SPARC as a therapeutic agent because of its various physiological activities and because it can be produced efficiently, economically, and in a large quantity.
It is another object of the invention to search and elucidate those substances which are involved in every change that occurs in neurons and synapses (such as proliferation, adhesion, migration, excitement) and to provide such substances as pharmaceutical compositions which are available for studying mechanisms of the nervous system and for prophylactic treatment of nervous diseases relating thereto.
As a result of intensive and extensive researches toward the solution of the above problems, the present inventors have found that by incorporating a gene encoding SPARC downstream of a gene encoding thioredoxin and expressing the resultant construct in E. coli, it is possible to produce SPARC as an easy-to-formulate, soluble protein in a large quantity without losing the physiological activities of SPARC.
Also, the present inventors have isolated and identified a group of genes the expression of which is specifically increased in the amygdala during chronic administration of morphine, and found that one of the genes is SPARC gene. Then, based on the above facts, the inventors have prepared a SPARC fusion protein and administered it together with morphine, and confirmed that morphine-induced spontaneous locomotor activity increases in the brain as a result of the administration. This means that synapse functions are changing. The inventors have also confirmed that the SPARC fusion protein exerts anti-cell adhesion activity and cell-rounding activity on nerve cells. Thus, the present invention has been achieved.
In the first aspect, the present invention relates to the following protein (a) or (b):
(a) a protein consisting of the amino acid sequence as shown in SEQ ID NO:2 of the Sequence Listing
(b) a protein which consists of the amino acid sequence as shown in SEQ ID NO:2 of the Sequence Listing having deletion, substitution or addition of one or several amino acids and which has physiological activities of SPARC.
The deletion, substitution or addition of amino acids can be introduced by site-specific mutagenesis, a technique well known in the art before the filing of the present application. The term xe2x80x9cone or several amino acidsxe2x80x9d means such a number of amino acids that can be deleted, substituted or added by site-specific mutagenesis.
The xe2x80x9cphysiological activities of SPARCxe2x80x9d mentioned above refers to angiogenesis, anti-cell adhesion activity (cell elongation inhibitory activity and cell-rounding activity), extracellular matrix remodeling (reduction of extracellular matrix production and enhancement of production of extracellular matrix-degrading proteases), bone formation (mineralization) and the like.
In the second aspect, the present invention relates to a DNA coding for the above-described protein, or a DNA consisting of a partial nucleotide sequence spanning from position 5209 to position 6609 of the nucleotide sequence as shown in SEQ ID NO:1 of the Sequence Listing.
In the third aspect, the present invention relates to a method for producing a SPARC fusion protein, comprising culturing in a medium a transformant obtainable by introducing into a host cell a recombinant vector incorporating the above-described DNA, allowing the host cell to produce and accumulate the protein encoded by the DNA in the resultant culture, and recovering the protein from the culture.
In the fourth aspect, the present invention relates to a pharmaceutical composition comprising the above-described protein.